Method of preparing subtilin



Patented May 23, 1950 2,508,378 nm'rnon or PREPARING sun'rmm Robert E.Feeney, El Cerrito, and John A. Garlbaldi, San Francisco, Calif.,assignors to the United States of America as represented by theSecretary of Agriculture No Drawing. Application February 12, 1948,Serial No. 8,017

6Claims.

(Granted under the act of March 3, 1883, as

amended April 30,

This application is made under the act of March 3, 1883, as amended bythe act of April 30, 1928, and the inventionherein described and claimedif patented'in any country, may be manufactured and used by or for theGovernment of the United States of America throughout the world forgovernmental purposes without the payment to us of any royalty thereon.

This invention relates to the production of the antibiotic subtilin byculturing a particular strain the invention.

subtilin is an antibiotic produced by a particular strain of Bacillussubtilis and has antibiotic activity against a number of pathogenicorganisms including Bacillus anthracis, Diplococcus pneumoniae,Neisseria gonorrheae, Mycobacterium tuberculosis, and Endamoebahistolytica.

Known methods of producing subtilin have not given uniform yields of theantibiotic because it was not realized heretofore that certain mineralelements play such an important part in the biosynthesis. It has beenfound that small amounts of these elements cause drastic differences inthe yield of subtilin. Thus the inventors have found that the followingelements are vital to the production of subtilin, i. e., potassium,magnesium, manganese, iron, zinc, sulphur, and phosphorus. Further, bycontrol of the amounts of these elements, maximum yields of theantibiotic may be obtained.

It is to be emphasized that control of these elements is not merely amatter of causing maximum cell growth. In many instances it is possibleto obtain good cell growth without obtaining a good yield of subtilin.For instance, rubidium may be substituted in the medium for potassium.In such case the cell growth is comparable to that with potassium butthe formation of subtilin is practically negligible. In the case ofmagnesium, an increase of from 2 P. P. M. of magnesium to '7 P. P. M. ofmagnesium will cause no significant change in the amount of cellsproduced yet the amount of subtilin will be increased more thaneight-fold by the same in-- crease in magnesium concentration. In thecase of zinc, an increase in the concentration of this 5 element from0.1 P. P. M. to 0.25 P. P. M. caused no significant change in the amountof cell growth yet the yield of subtilin was more than doubled by theincrease in zinc concentration.

The amounts of the elements necessary to obtain good yields of subtilinare as follows:

Potassium-at least 60 P. P. M., preferably at least 100 P. P..M. Thesefigures represent only the lower limits of concentration. This elementdoes not exhibit a toxic effect at higher concentrations. Generally, aconcentration of about 400 to 1000 P. P. M. is used to insure presenceof suflicient potassium.

Magnesium-about 4 P. P. M. to about 300 P. P. M., preferably 7 to 50 P.P. M. If the medium does not contain citric acid or other organichydroxy-acid (or salt thereof), magnesium has a deleterious effect onthe yield of subtili'n if present in a concentration higher than about50 P P. M.

Where the medium contains citric acid or other organic acids (or saltthereof) this deleterious effect is counteracted and the concentrationof magnesium may be as high as about 300 P. P. M.

Manganese-0.4 P. P. M. to 100 P. P. M., preferably l P. P. M. to 100 P.P. M.

Iron0.6 to about 100 P. P. M., preferably about 1.5 to P. P. M. If themedium does not contain citric acid or other organic hydroxy-acid (orsalt thereof), iron has a deleterious eflect on the yield of subtilin ifpresent in a concentration higher than about 8 P. P. M. Where the mediumcontains citric acid or other organic oxy acid (or salt thereof) thiseffect is counteracted and the concentration of iron may be as high asabout 100 P. P. M.

Sulphur-at least P. P. M., preferably at least '70 P. P. M. The amountof sulphur may be increased to any desired degree above these lowerlimits without decreasing the yield of subtilin. Generally aboutSOD-1,000 P. P. M. are used to insure sufficient sulphur in the medium.

Phosphorus-at least 50 P. P. M., preferably at least 100 P. P. M. Wherethe amount of phosphorus is greater than about 200 P. P. M., citric acid(or a salt thereof) may be added to'the medium. Where such precaution istaken, the amount of phosphorus may be increased to any desired levelwithout deleterious eflect. In gen- 66 eral, a concentration of about1000 P. P. M. is

Sucrose 100 g.

Nazsos 4 g.

NaCl 0.3 g.

Salt mixture 100 ml.

(NHt) M04 8 g.

l-Asparagin 2g.

Glutamic acid 2 g.

Redistilled water Sufilcient to give 1 liter of medium The salt mixturecontained the following salts per liter: K01, 3.81 MgC12'6H2O, 4.18 g.;ZnClz, 0.104 g.; FeCla-GHzO, 0.245 g.; NmC12-4H2O, 0.181 g. When addedto the medium in the amount indicated, it gave (in P. P. M.) K, 200; Mg,50; Zn, 5.0; Fe, 5.0; and Mn, 5.0.

The medium was adjusted to pH of 6.8-6.9 with dilute sodium hydroxideand sterilized before use.

The culture employed in the experiments described in the examples was astrain of B. subtz'lz's designated in the stock culture collection ofthe U. S. Department of Agriculture, Northern Regional ResearchLaboratory, as B-543.

Subtilin was assayed by a short incubation period turbidimetricbacteriostatic method similar to that described by McMahan [Jour. Biol.Chem, vol. 153, p. 249 (1944)] for penicillin. In this assay, thesamples of culture Were assayed against a standard sample of isolatedsubtilin using Micrococcus conalomeratus as the test organism. Subtilinyields are expressed in milligrams per liter of culture medium asdetermined against this standard. The standard used in these experimentswas the same as that employed in the applications of Stubbs et al., Ser.No. 776,397, filed Sept. 26, 1947: Dimick et al., Ser. No. 777,843,filed Oct. 3, 1947, now Patent No. 2,476,085, and Dimick et al.,776,396, filed Sept. 26, 1947, now Patent No. 2,459,139.

EXAMPLE I.-EF'FECT OF POTASSIUM In this example, the medium used wasthat described above with potassium omitted from the salt mixture.Varying amounts of potassium were added, as KCl, to indicate the effectof this element. In each case a 50 ml. sample of the medium wasinoculated with B. sultilis and the culturing carried out by a surfacetechnique at a temperature of C. After incubation for '12 hours theculture was assayed for subtilin activity and the dry weight of thepellicle determined as a measurement of the degree of cell growth. Thefollowing results were obtained:

4 None of the three elements closest related chemically to potassium arecapable of replacing this element. Thus neither sodium nor caesiumsupports growth in the absence of potassium. Rubidium can quantitativelyreplace potassium mole per mole'as far as growth is concerned but cannot replace potassium for the production of subtilin. Thus when anequivalent amount of rubidium was substituted for potassium inexperiment No. 3, the yield of pellicle was 210 mg. but the subtilinyield was less than 20 mg./l. When experiment No. 7 was repeated usingan equivalent amount of rubidium, the yield of pellicle was 420 mg. andthe yield of subtilin 72 mg./l.

EXAMPLE 1I.EFFECT OF MAGNESIUM In this example, the medium used was thatdescribed above with magnesium omitted from the salt mixture. Varyingamounts of magnesium were added, as MgCl2'6H2O, to indicate the effectof this element. In each case a 50 ml. sample of the mediumWasinoculated with B. subtilis and the culturing carried out by aSurface technique at a temperature of 35 C. for 72 hours. The followingresults were obtained:

Table 2 Magnesium D rywei ht Subtilm 23x35 or pellic e, produced, P. I,mg. mgJlitcr It was found by experimentation that calcium and beryllium,the elements closest related chemically to magnesium cannot besubstituted for this element. Calcium, tested at concentrations of 1, 5,10, and 100 P. P. M., or beryllium, tested at 0.3, 1.0, and 3.5 P. P. M.gave no increase in yield over the very slight yield obtained in theabsence of added magnesium.

EXAIWPLE III.EFFECT OF MANGANESE Table 3 Manganese D rywei ht Subtilinfig g oi pelli e, produced P. r. M.

By experimentation it was found that none of the following elements arecapable of being substituted for manganese, i. e., cadmium, gallium,aluminum, nickel, tin, thallium, cobalt, molybdenum, boron, barium,lead, mercury, strontium,

46 and chromium.

mun: rv.-mcr O1" IRON r. r. 11. mg. mgJiiter' o 40 o 0.: 150 so 1.25 muse 2.0 50 6!) 9 t 276 000 It was iound by experimentation that noneoithe following elements are capable of being substituted for iron,i.'e., cadmium, gallium, aluminum, barium, strontium, coppennickei, tin,co-' bait, molybdenum, calcium. lead, and thallium.

7 EXAMPLE v.- -m'sc'r or zmc A medium which had the iollowingingredients was prepared:

Sucrose 100 g. Asparagin 2.0 g. Glutamic acid 2.0 g. 'Iryptophane 0.1 g.NaeSOr 4.0 g. (NHDsHPOs 1 8.0 8. NaCl 0.3 3. Extract of Steii'ens wasteliquor 100 ml. Water --Sufllcient to make 1 liter The medium alsocontained the following salts in the amounts indicated per liter oimedium: MgSOHIHzO, 0.51 3.; C8Cl2, 0.14 g.; FeCh-BHzO. 0.019 g.; KCl,0.095 3,; and Much-411:0, 0.18 g.

This medium was extracted several times with a carbon tetrachloridesolution 01' diphenylthiocarbazone to remove traces oi zinc. To themedium was then added graded amounts of zinc, as ZnCla, to show theeflect of this element. In each case a 50 mi. sample oi the medium wasinoculated with B. subtilis and the culturing carried out by a suriacetechnique at a temperature of 35 for 64 hours. The following resultswere obtained:

Table Zinc added Dry weight Bubtilin proto medium, of pcllicle, duced.mgJ P. P. M. mg. liter 0 83 16 us 100 an l Iifl 610 no can 6 thallium.lead. copper. cobalt. m mium. and mercury.

murm- WW or mosrnonus Amediumwaspreparedoontainingtheiollowingingredients:

Sucrose g. Nam 4 3. NaCl 0.3 g. Citric acid 6 g.

Glutamic acid 8 g. Bait mixture 100 ml. water Bumcient to make 1 l.

The salt mixture contained the iollcwing salts per liter: RC1. 3.81 8.;MGCh'GI-BO. 4.18 8.; 211612. 0.104 g.; Feels-611:0, 0.245 g.; Much-48:0,0.181 g. when added to the medium in the amount indicated it gave (in P.P. M.): potassium. 200; magnesium, 50: zinc, 5; iron, 5.0; andmanganese, 5.

Varying amounts oi phosphorus were added (as disodium phosphate) toindicate the eii'ect of this element. In each case a 50 ml. sample ofthe medium was inoculated with B. subtilis and the culturing carried outby a suriace technique at a temperature oi 35 0. ior 60 hours. 'Iheiollowing results were obtained:

Table 6 Dry we ht Subtilin c! pelli e, duced. mg./60 ml. liter VHF-maror summm A medium was prepared containing the iollow- The salt mixturewas the same as that em ployed in the preeeding'example.

Varying amounts "of sulphur were added, as

sodium sulphate, to indicate the efiect oi this element. In each case,a, 50 ml.sample oi the medium was inoculated with B. subiilis and theculturing carried out by a suriace technique at a temperature of 35 C.ior 72 hours. The iollowing results were obtained:

Table 7 Drywe m Subtilin $3 2 oi pelii e, produced, P. P. M.

0 4o 0 so 400 soo an 460 see Sucrose 7 mm VIII A medium was made up asiollowsz 33 lbs.

*NaaSOr 600 g.

'. Yeast extract 750 g. Diammonium phosphate 637 g. Citric acid 1755 g.

* Napl 45 g. Ammonium hydroxide, 28% 1.8 1.

Water Sufilcient to make 150 liters Mineral salts were added to furnish:

. million Zinc, iron, manganese, each Magnesium 50 .Potassium 400 Themedium was introduced into a large size i'ermentor, sterilized withsteam at lb. pressure,

then inoculated with a culture of B. subtilis grown on asparagus juicemedium. The culturing was accomplished under submerged conditions byagitating the culture and pumping air into it. After allowing the growthto proceed for 10 hours at C., the culture was harvested and 135 gramsof subtilin was obtained (900 mg. per liter).

' EXAMPLE IX The medium in this case was similar to that used in ExampleVIII with the exception that the sodium chloride was omitted and theconcentration'of zinc, iron, manganese, and magnesium I weredoubled,also 830 ml. of asparagus juice were added. This medium was sterilized,inoculated,

and the growth carried out as in the previous example. The yield ofsubtilin in 10 hours was 189 "grams or 1260 mg. per; liter.

We have emphasized above the mineral re- Q quireinents oi the medium. Itis, of course, ob-

vious that the medium must contain the proper ingredients to supportgrowth of the bacillus.

Thus the medium must contain, besides water, (a) a source of energy, (b)a source of nitrogen,

and (c) the elements specified above. The source of energy maybe acarbohydrate such as sucrose, glucose, fructose, galactose, xylose,mannitol, glycerol, maltose, starch, etc. It is generally preferred touse sucrose as this sugar gives high yields of subtilin. Good yields arealsoobtained with glycerol or glucose. The concentration of -thecarbohydrate should be enough to provide sufllcient nutriment for propergrowth. In the case of sucrose good results are obtained with moniumacetate, etc. Nitrates may also be used, for example, sodium nitrate,ammonium nitrate, potassium nitrate, etc. In general the concentrationof nitrogen should be about .075% to 1%. No deleterious effect is causedby adding more than 1%.

With regard to the elements potassium, magnesium, manganese, iron, andzinc, these elements may be supplied to the medium by any of their saltswhich are soluble enough to give the desired concentration. Thus thenitrates, sulphates, acetates, chlorides of these metals can beutilized. It is immaterial what salt is used as long as it makesavailable these metal ions.

proteins,-yeast extract, glutamic acid, aspartic acid, asparagin.ryptophane, threonine, histidine, tyrosine, cystine, lanthionine, orother amino acids. For the sake of economy it is usually preierable touse an inorganic nitrogen compound such as ammonia or salts thereof suchas ammonium phosphate,"'ammoni1im citrate, am-j In the case ofphosphorus and sulphur, these elements can be supplied by suitablewater-soluble salts such as sodium phosphates, sodium sulphate, sodiumphosphites, sodium sulphite, etc. Obviously other metal ions can be usedin place of sodium, such as potassium, ammonium, etc.

The addition of citric acid or other organic acid (or a water-solublesalt thereof such as sodium, potassium or salt) to the medium is oftenbeneficial to act as a buffer and to keep the various salts in solution.Citric acid is especially beneficial where the concentration of iron,magnesium, or phosphorus is too high due, for example, to impurities inthe materials used. Thus if the amount of iron in the medium is found toexceed 8 P. P. M. then addition of citric acid will render the mediumsuitable for maximum growth. The concentration of citric acid may bevaried. depending upon the excessive concentration of elements, fromabout .05% to about 1%. Calcium is often present in natural sources ofenergy such as molasses, cereal extracts, asparagus juice, etc., and itsdeleterious efiect can be counteracted by adding citric acid. Instead ofusing pure citric acid one can use natural materials containing the sameor similar acids such as asparagus juice, alfalfa juice, beet molasses,Steflens waste liquor, and other agricultural materials of this type.

Subtilin can be produced in a continuous manher by proceeding in thefollowing way: A me-- dium is prepared containing water, the minerals(K, Mg, Mn, Fe, Zn, S, and P) in the concentrations herein set forth,citric acid or similar organic acid in the proportions set forth, and asmall amount of a source energy (such as sucrose, glucose, or glycerol)and a small amount of a source of nitrogen (an ammonium salt ornitrate). The sources of energy and nitrogen are added only insufficient quantity to enable initial growth to proceed. The medium isinoculated with the' proper strain of B. subtilis and culturing isstarted under the conditions herein set forth. Then, as the culturingproceeds, additional source of energy and source of nitrogen are addedas required for the growth of the organism and production oi subtilin. Aconvenient method of adding the source of nitrogen is. to add gaseousammonia directly to the culture. Part of the culture may be drawn offfrom time to time for isolation of subtilin.

' The pH of the culture may vary from about 5.5

to about 7.5. Preferably, the pH is adjusted to about from 6.5 to 7.0 atthe start of the incubation, and no attempt need be made thereafter tomaintain the pH constant during the production cycle.

The temperature of the submerged culture can be varied from about 30 C.to about 40 C., best results generally being obtained at about 35 C. Attemperatures higher than about 35 0., subtilin is produced at. a fasterrate, but the final 9 yield of subtiiin decreases markedly, while attemperatures below about 35 C., the production of subtilin is slow, andthus the operation must be continued for a longer period of time toobtain maximum yield of subtilin. A convenient method of obtaining ahigh yield of subtilin in a shortened period of time is by employing atemperature above 35 C. (up to about 40C.) durin the initial period ofgrowth, and then decreasing the temperature to, about 35 C. when maximumcell volume is obtained. In this manner, the rate of multiplication ofthe cells is enhanced by the increased temperature, and the productionor subtilin. which lags behind the cell production is enhanced by thelower temperature maintained after maximum cell formation has beenreached. During the period of rapid growth oi the cells, the process isexothermic, and adequate cooling means should be employed to prevent thetemperature from rising considerably above 40 C.

The subtilin can be produced by a surface technique or by an aerobic,submerged technique, the latter being preferred to prepare large amountsof the antibiotic. Suitable methods of manipulation and other factors insubmerged culture are disclosed in the patent application of Stubbs etal., Ser. No. 776,397, filed Sept. 26, 1947. Methods of isolating theantibiotic from the culture are disclosed in the patent application ofDimick et al., referred to above.

The abbreviation "P. P. M." used herein stands for parts per million.

In broad aspect, the process is characterized as one for preparingsubtilin comprising inoculating a nutrient medium with asubtiiin-producing strain of Bacillus subtilis and incubating theresulting culture untila substantial amount of subtilin is produced,said medium containing a source of energy, a source of nitrogen, andmineral elements in the following concentration:

Parts per million K At least 60 Mg 4 to 300 Mn 0.4 to 100 r 0.6 to 100Zn 1 to 50 S At least 20 P At least 50 Having thus described ourinvention, we claim:

1. A process for preparing subtilin comprising inoculating a nutrientmedium with a subtilinproducing strain of Bacillus subtilis andincubating the resulting culture untfl a substantial amount of subtilinis produced, said medium containing a source of energy, a source ofnitrogen,

and mineral elements in the following concentration:

Partspermiliion Atleast100 5to50 1to50 1.25to8 1to10 AtieastflAtieast200 amount of subtilin is produced, said medium containing asource of energy, a source of nitrogen, about 0.05 to about 1% citricacid, and mineral elements in the following concentration:

Partspermlllion At least 5 tooo 1 to 50 1.25to 1o 1 to 10 At leastoo At1east200 6. The process of claim 1 in which the magnesium is about 5 to"I P. P. M. and the phosphorus content is 200 to 1000 P. P. M.

ROBERT E. FEENEY. JOHN A. GARIBAIJJL REFERENCES CITED The followingreferences are of record the file of this patent:

UNITED STATES PATENTS Name Date Burkholder Nov. 21, 1944 OTHERREFERENCES Henrici: Biology of Bacteria (1934), D. 0. Heath a. Ca, page218.

Jansen et al.: Arch. Biochem. (1944), 4. pos s Feeney et al.: Arch. ofBiochem., Oct. 1947, pages 13 to 17.

'smglgggpq Number

1. A PROCESS FOR PREPARING SUBTILIN COMPRISING INOCULATING A NUTRIENTMEDIUM WITH A SUBTILINPRODUCING STRAIN OF BACILLUS SUBTILIS ANDINCUBATING THE RESULTING CULTURE UNTIL A SUBSTANTIAL AMOUNT OF SUBTILINIS PRODUCED, SAID MEDIUM CONTAINING A SOURCE OF ENERGY, A SOURCE OFNITROGEN, AND MINERAL ELEMENTS IN THE FOLLOWING CONCENTRATION: